The basic purpose of drug metabolism in the body is to make drugs more water soluble and thus more readily excreted in the urine or bile. One common way of metabolizing drugs involves the alteration of functional groups on the parent molecule (e.g. oxidation), via the cytochrome P450 enzymes. These enzymes are predominantly found in the liver. Cytochrome p450 enzymes are involved in numerous drug metabolism pathways as well as pathways used to make cholesterol, steroids, and other important lipids such as prostacyclins and thromboxane A2. Many drug interactions are a result of inhibition or induction of cytochrome P450 enzymes.
Mammalian cytochrome P450 genes encode a superfamily of hemeproteins that are active in the oxidative metabolism of endogenous and exogenous compounds. Cytochrome P450 (referred to as CYP) are now classified into families on the basis of amino acid similarity; within families cytochrome P450 exhibit >40% similarity and >55% similarity within subfamilies. The cytochrome P450 enzymes are designated by the letters “CYP” followed by a numeral, a letter and another numeral (e.g. CYP2D6). In humans there are more than 20 different CYP enzymes. According to a recent compilation, the cytochrome P450 2C subfamily contains 36 distinct genes and pseudogenes, and is the largest cytochrome P450 subfamily that has been identified to date. It is generally accepted that mammalian cytochrome P450 genes are regulated primarily at the level of transcription, and there is little information known on the factors that regulate transcription.
There are a wide variety of polymorphisms (e.g. mutations) in p450 enzymes, and many of these polymorphisms result in (or are associated with) the inhibition or induction of enzymatic activity. These polymorphisms frequently occur in ethnic populations. The cytochrome P450 genes that have been studied most intensively in this regard are cytochrome P450 2D6, the debrisoquine hydroxylase gene, and cytochrome P450 2C19, which codes for the S-mephenyloin hydroxylase. A variety of other P450 enzymes exhibit variation in expression levels; for example, cytochrome P450 3A4, the major cytochrome P450 present in human liver, varies over a several-fold range at both the protein and mRNA levels. In addition, many of the P450 enzymes are subject to induction by different drugs, including barbiturates, antibiotics, etc.
Of particular interest to the present invention are CYP1A1, CYP1A2, CYP1B1, CYP2C19, CYP3D6, CYP2E1 and CYP3A4. CYP2D6 has been studied extensively because it exhibits significant diversity, with roughly 7 to 10 percent of Caucasians being poor metabolizers of drugs metabolized by CYP2D6. Patients with normal CYP2D6 activity are termed extensive metabolizers, with Asians and African Americans being less likely than Caucasians to be poor metabolizers. Poor metabolizers are at risk for drug accumulation and toxicity from drugs metabolized by this enzyme. While only 2 to 6 percent of total liver cytochrome P450 is CYP2D6, nearly 25 percent of clinically useful medications are metabolized by this enzyme. Poor metabolizers of CYP2D6 substrates are at risk for increased toxicity from medications that are metabolized by CYP2D6. Conversely, when formation of an active metabolite is essential for drug action, poor metabolizers of CYP2D6 can exhibit less response to drug therapy compared with extensive metabolizers. About 15 percent of clinically used medications are metabolized by CYP1A2, and it is the only CYP that is induced by tobacco and other polycyclic aromatic hydrocarbons.
CYP2E1 metabolizes a relatively small fraction of medications (although it has a significant role in the metabolism of acetaminophen), it plays a significant role in activation and inactivation of toxins. It is inducible by ethanol and metabolized primarily small organic molecules.
Members of the CYP3A family are the most abundant and most clinically significant cytochrome enzymes in humans, with CYP3A4 being the most common form and the most widely implicated in most drug interactions. The CYP3A family is located in the small intestine and in addition to drug, is also responsible for metabolizing most of the body's endogenous steroids.
CYP2C19, along with CYP2D6, also exhibits genetic polymorphism, with 3 percent of Caucasians and 20 percent of Japanese lacking the enzyme completely. These individuals are at risk for more frequent and more severe adverse effects because of decreased elimination of drugs metabolized by CYP2C19.
Thus, detection of specific P450 SNPs is important in diagnostic medicine and molecular biology research and also, to understand the mechanism of action of many drugs and are likely to be the direct cause of therapeutically relevant phenotypic variants and/or disease predispositions.
Accurate SNP detection requires good sensitivity in the SBE assays. Two major hurdles for highly parallel screening of SNPs on microarrays are: 1) the necessity to amplify DNA regions spanning the SNPs by PCR to achieve sufficient sensitivity and specificity of detecting a single-base variation in the complex human genome in a reproducible way; and, 2) the ability to distinguish unequivocally between homozygous and heterozygous allelic variants in the diploid human genome. Differential hybridization with allele-specific oligonucleotide (ASO) probes is most commonly used in the microarray format (Pastinen et al., Genome Research 2000). The requirement for sensitivity (i.e. low detection limits) has been greatly alleviated by the development of the polymerase chain reaction (PCR) and other amplification technologies which allow researchers to amplify exponentially a specific nucleic acid sequence before analysis (for a review, see Abramson et al., Current Opinion in Biotechnology, 4:41-47 (1993)). Multiplex PCR amplification of SNP loci with subsequent hybridization to oligonucleotide arrays has been shown to be an accurate and reliable method of simultaneously genotyping at least hundreds of SNPs; see Wang et al., Science, 280:1077 (1998); see also Schafer et al., Nature Biotechnology 16:33-39 (1998).
Specificity, in contrast, remains a problem in many currently available gene probe assays. The extent of molecular complementarity between probe and target defines the specificity of the interaction. Variations in the concentrations of probes, of targets and of salts in the hybridization medium, in the reaction temperature, and in the length of the probe may alter or influence the specificity of the probe/target interaction.
It may be possible under some circumstances to distinguish targets with perfect complementarity from targets with mismatches, although this is generally very difficult using traditional technology, since small variations in the reaction conditions will alter the hybridization. New experimental techniques for mismatch detection with standard probes, as defined in greater detail below, include, but are not limited to, OLA, RCA, Invader™, single base extension (SBE) methods, allelic PCR, and competitive probe analysis. In SBE assays, a polynucleotide probe is attached to a support and hybridized to target DNA.
Generally, for SBE assays, probe sets are designed such that the nucleotide at the 3′ end of the probe is either matched or mismatched with the queried base in the target. If the base matches and hybridizes, the DNA polymerase will extend the probe by one base in the presence of four labeled-terminator nucleotides. Alternately, if the 3′ base is mismatched, the DNA polymerase does not extend the probe. Thus, the identity of the SNP or queried base in the target is determined by the probe set that is extended by the DNA polymerase.
Some probes form internal stem-loop structures resulting in target-independent self-extension of the probe thus giving a false positive signal that interferes with determination of the SNP base. The present invention aims to overcome such problems.
Accordingly, it is an object of the present invention to provide compositions and methods for evaluating samples from one or more patients, to ascertain the level and/or genotype of various P450 enzymes present. Another object of the present invention is to increase the sensitivity and specificity of the P450 SBE assays. The present invention uses a combination of amplification methods, and, a variety of methods to prevent self-extension of capture probes in the absence of target thereby reducing the occurrence of false positive results.